This invention relates to vascular therapy.
Angiogenesis results from endothelial cell proliferation induced by angiogenic factors. Angiogenic factors bind to receptors on endothelial cells which line blood vessels. This event triggers signals which cause the cells to proliferate; the proliferating endothelial cells secrete proteases which digest the basement membrane surrounding a vessel. The junctions between the endothelial cells are altered, allowing projections from the cells to pass through the space created. These outgrowths then become new blood vessels, e.g., capillaries.
Vascular endothelial cell growth factor (VEGF) and VEGF receptors (VEGF-Rs) play a role in vasculogenesis and angiogenesis. Although VEGF is secreted by a variety of cell types, including vascular smooth muscle cells, osteoblasts, fibroblasts, and macrophages, its proliferative and chemotactic activities are restricted to endothelial cells. VEGF signaling is mediated by two VEGF-Rs, the endothelial cell-specific tyrosine kinase receptors, flt-1 and KDR/flk-1. Despite its importance in VEGF signaling, the molecular mechanisms of VEGF and VEGF-R expression have not been elucidated.
The invention is based on the discovery that endothelial PAS domain protein-1 (EPAS1) binds to cis-acting regulatory sequences associated with genes encoding such angiogenic factors as VEGF and VEGF-Rs such as KDR/flk-1 and flt-1, thereby transactivating the promoters of such genes. Accordingly, the invention features a method of increasing the level of EPAS1 in a cell, e.g., an endothelial cell. An increase in the level of EPAS1 leads to increased promoter transactivation and increased transcription of genes encoding angiogenic factors which participate in the blood vessel formation.
The invention also includes a novel basic helix-loop-helix/Per-AhR-Arnt-Sim (bHLH/PAS) protein which binds to EPAS1 and forms a heterodimer which transactivates transcription of genes encoding angiogenic factors. Increasing the level of ARNT4 in a cell, e.g., an endothelial cell also leads to increased promoter transactivation and increased expression of angiogenic factors which participate in the blood vessel formation.
Angiogenic factors are proteins or polypeptides and ligands thereof that participate in the process of new blood vessel formation. For example, angiogenic factors include VEGF, VEGF-Rs, and other signalling proteins such as intracellular tyrosine kinases which participate in the angiogenic process. Preferably, the angiogenic factors are expressed in endothelial cells, e.g., VEGF, VEGF-Rs such as KDR/flk-1 or flt-1, and tyrosine kinases such as Tie2.
A method of inhibiting angiogenesis in a mammal is carried out by administering to the mammal a compound which inhibits binding of EPAS1 to cis-acting transcription regulatory DNA associated with a gene encoding an angiogenic factor. Angiogenesis is also inhibited by administering a compound which inhibits binding of EPAS1 to ARNT4, i.e., a compound which inhibits the formation of a functional heterodimer that can transactivate a promoter of gene encoding an angiogenic factor. The angiogenic factor is preferably VEGF, a VEGF-R such as KDR/flk-1 or flt-1. For example, the compound inhibits transcription of the angiogenic factor by binding to a cis-acting regulatory sequence such as the sequence 5xe2x80x2 GCCCTACGTGCTGTCTCA 3xe2x80x2 (SEQ ID NO:1) in VEGF promoter DNA. For example, the compound is an EPAS1 polypeptide that binds to a cis-acting regulatory sequence of a gene but fails to transactivate the promoter of the gene, e.g, a polypeptide lacking a transactivation domain (amino acids 486-690 of EPAS1).
Table 1: Transactivation Domain of Human EPAS1 EDYYTSLDNDLKIEVIEKLFAMDTEAKDQCSTQTDFNELDLETLAPYIPMDGEDFQLSPI CPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKKTEPEHRPMS SIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQWPPDPPLHFGPTKWAVGDQRTEFLGAA PLGPPVSPPHVSTFKTRSAKGFGAR (SEQ ID NO:2)
When such an EPAS1 mutant is bound to a cis-acting regulatory DNA, it prevents wild type EPAS1 binding and thereby inhibits transcription of a gene encoding an angiogenic factor (and, in turn, angiogenesis). For example, the EPAS1 polypeptide contains the N-terminal binding domain (amino acids 14-67 of EPAS1; RRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIMRLEISFLRTH; SEQ ID NO:3) more preferably the EPAS polypeptide contains amino acids 1-485 of human EPAS1. The amino acid sequence of such an EPAS1 dominant negative mutant polypeptide and DNA encoding such a mutant polypeptide is provided below.
Rather than administering EPAS1 polypeptides or ARNT4 polypeptides, the method may be carried out by administering DNA encoding such polypeptides. For example, the compound is a nucleic acid encoding an EPAS1 polypeptide lacking amino acids 486-690 of EPAS1. For example, the nucleic acid encodes a dominant negative mutant of EPAS1 which contains amino acids 1-485 of wild type EPAS1, i.e., SEQ ID NO:5.
For antisense therapy, the compound is a antisense nucleic acid molecule containing at least 10 nucleotides the sequence of which is complementary to an mRNA encoding all or part of a wild type EPAS1 polypeptide. Preferably, the compound, e.g., an antisense oligonucleotide or antisense RNA produced from an antisense template, inhibits EPAS1 expression. For example, the compound may inhibit EPAS1 expression by inhibiting translation of EPAS1 mRNA. For example, antisense therapy is carried out by administering a single stranded nucleic acid complementary at least a portion of EPAS1 MRNA to interfere with the translation of MRNA into protein, thus reducing the amount of functional EPAS1 produced in the cell. A reduction in the amount of functional transactivating EPAS1 reduces the level of transcription of angiogenic factors such as VEGF or VEGF-Rs, resulting in a decrease in new blood vessel formation.
Alternatively, the compound is an EPAS1-specific intrabody, i.e., a recombinant single chain EPAS1-specific antibody that is expressed inside a target cell, e.g., a vascular endothelial cell. Such an intrabody binds to endogenous intracellular EPAS1 and prevents it from binding to its target cis-acting regulatory sequence in the promoter region of a gene encoding an angiogenic factor such as VEGF or a VEGF-R. An ARNT4-specific intrabody is also useful to inhibit angiogenesis.
Angiogenesis contributes to the progression of atherosclerotic lesions. Thus, compounds are administered to a site of an atherosclerotic lesion in a mammal to inhibit growth of a lesion. Compounds may also be locally administered to a tumor site to reduce blood vessel formation, thereby depriving a tumor of blood supply and inhibiting tumor growth. VEGF itself is a growth factor for some tumors; the methods described above directly inhibit VEGF expression, and thus, are particularly useful for treating such tumor types.
The invention also includes an antibody which binds to EPAS1. The antibody preferably binds to the C-terminal portion of EPAS1 (e.g., a polypeptide having the amino acid of SEQ ID NO:17 or 18). The antibody is a polyclonal or monoclonal antibody which specifically binds to the EPAS1. Preferably, the antibody binds to an epitope within the C-terminal transactivation domain (SEQ ID NO:2). The invention encompasses not only an intact monoclonal antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab)2 fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin.
To promote angiogenesis in a mammal, a compound, e.g., DNA encoding EPAS1 or a functional fragment thereof, which increases expression of VEGF or a VEGF-R in an endothelial cell is administered to a mammal, e.g., an adult mammal which has been identified as being in need of therapy to promote angiogenesis such as a patient suffering from peripheral vascular disease. A functional fragment of EPAS1 is one which binds to DNA in the promoter region of a gene encoding an angiogenic factor.
The invention also features an EPAS-binding element, ARNT4 and a nucleic acid which encodes ARNT4. For example, the nucleic acid includes a sequence which encodes the amino acid sequence a naturally-occurring human ARNT4 (SEQ ID NO:19). The DNA may encode a naturally-occurring mammalian ARNT4 polypeptide such as a human, rat, mouse, guinea pig, hamster, dog, cat, pig, cow, goat, sheep, horse, monkey, or ape ARNT4. Preferably, the DNA encodes a human ARNT4 polypeptide, e.g., a polypeptide which contains part or all of the amino acid sequence of SEQ ID NO:19. The invention includes degenerate variants of the human cDNA (SEQ ID NO:20). The DNA contains a nucleotide sequence having at least 50% sequence identity to SEQ ID NO:20. For example, the DNA contains a sequence which encodes a human ARNT4 polypeptide, such as the coding sequence of SEQ ID NO:20 (nucleotides 220 to 2025 of SEQ ID NO:20). The DNA contains a strand which hybridizes at high stringency to a strand of DNA having the sequence of SEQ ID NO:20, or the complement thereof. The DNA has at least 50% sequence identity to SEQ ID NO:20 and encodes a polypeptide having the biological activity of a ARNT4 polypeptide, e.g. the ability to bind to EPAS1 to form a heterodimer. Preferably, the DNA has at least 75% identity, more preferably 85% identity, more preferably 90% identity, more preferably 95% identity, more preferably 99% identity, and most preferably 100% identity to the coding sequence of SEQ ID NO:20.
Nucleotide and amino acid comparisons are carried out using the CLUSTAL W sequence alignment system with (Thompson et al., 1994, Nucleic Acids Research 22:4673-4680 or http://www.infobiogen.fr/docs/ClustalW/clustalw.html). Amino acid sequences were compared using CLUSTAL W with the PAM250 residue weight table. xe2x80x9cPer cent sequence identityxe2x80x9d, as that term is used herein, is determined using the CLUSTAL W sequence alignment system referenced above, with the parameters described herein. In the case of polypeptide sequences which are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Hybridization is carried out using standard techniques, such as those described in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, 1989). xe2x80x9cHigh stringencyxe2x80x9d refers to nucleic acid hybridization and wash conditions characterized by high temperature and low salt concentration: wash conditions of 65xc2x0 C. at a salt concentration of 0.1xc3x97SSC. xe2x80x9cLowxe2x80x9d to xe2x80x9cmoderatexe2x80x9d stringency denotes DNA hybridization and wash conditions characterized by low temperature and high salt concentration: wash conditions of less than 60xc2x0 C. at a salt concentration of 1.0xc3x97SSC. For example, high stringency conditions include hybridization at 42xc2x0 C., and 50% formamide; a first wash at 65xc2x0 C., 2xc3x97SSC, and 1% SDS; followed by a second wash at 65xc2x0 C. and 0.1%xc3x97SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to an ARNT4 gene are detected by, for example, hybridization at 42xc2x0 C. in the absence of formamide; a first wash at 42xc2x0 C., 6xc3x97SSC, and 1% SDS; and a second wash at 50xc2x0 C., 6xc3x97SSC, and 1% SDS.
A vector containing an ARNT4-encoding DNA is also within the invention. Preferably the DNA which includes an ARNT4-encoding DNA is less than 5 kilobases in length; more preferably, the DNA is less than 4 kilobases in length, more preferably the DNA is less than 3 kilobases in length, and most preferably the DNA is approximately 2 kilobases or less in length. The invention also provides a method of directing cardiac-specific or smooth muscle cell-specific expression of a protein by introducing into a cell an isolated DNA containing a sequence encoding the protein operably linked to the tissue-specific promoter. A cell containing the DNA or vector of the invention is also within the invention.
By xe2x80x9csubstantially pure DNAxe2x80x9d is meant DNA that has a naturally-occurring sequence or that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the ARNT4 gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a procaryote or eucaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Also within the invention is a substantially pure human ARNT4 polypeptide. The term ARNT4 polypeptide includes a polypeptide having the amino acid sequence and length of the naturally-occurring ARNT4 as well as fragments of the full-length naturally-occurring ARNT4. The polypeptide contains the amino acid sequence of SEQ ID NO:19. Preferably the polypeptide contains an amino acid sequence which is at least 50% identical to SEQ ID NO:19. Preferably, the amino acid sequence has at least 75% identity, more preferably 85% identity, more preferably 90% identity, more preferably 95% identity, more preferably 99% identity, and most preferably 100% identity to the amino acid sequence of SEQ ID NO:19. For example, the ARNT4 polypeptide may have the amino acid sequence of the naturally-occurring human polypeptide, e.g., a polypeptide which includes the amino acid sequence of SEQ ID NO:19. The invention also encompasses a polypeptide with the amino acid sequence of a segment of SEQ ID NO: 17 which spans residues 75 to 128, inclusive, or a segment spanning residues 155 to 207, inclusive, of SEQ ID NO:19, or a segment spanning residues 232 to 384 of SEQ ID NO:19. Preferably, such a polypeptide has a biological activity of a naturally-occurring ARNT4 polypeptide, e.g, heterodimer formation with EPAS1 or the ability to transactivate transcription under the control of a VEGF promoter.
A substantially pure ARNT4 polypeptide is obtained by extraction from a natural source; by expression of a recombinant nucleic acid encoding a ARNT4 polypeptide; or by chemically synthesizing the protein. A polypeptide or protein is substantially pure when it is separated from those contaminants which accompany it in its natural state (proteins and other naturally-occurring organic molecules). Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, ARNT4. Purity is measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eucaryote but produced in E. coli or another procaryote, or in a eucaryote other than that from which the polypeptide was originally derived.
The invention also includes a transgenic non-human mammal, the germ cells and somatic cells of which contain a null mutation in a gene encoding an ARNT4 polypeptide. For example, the null mutation is a deletion of part or all of an exon of ARNT4. Preferably, the mammal is a rodent such as a mouse. An antibody which specifically binds to a ARNT4 polypeptide is also within the invention.
Angiogenesis is inhibited by administering to a mammal a compound which inhibits binding of EPAS1 to ARNT4 such as an ARNT4 polypeptide. For example, the compound is a polypeptide or peptide mimetic which contains the amino acid sequence of residues 75 to 128, inclusive, of SEQ ID NO:19, the amino acid sequence of residues 155 to 207, inclusive, of SEQ ID NO:19, or a the amino acid sequence of residues 232 to 384 of SEQ ID NO:19.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.